OmpF Synthesis Research Articles

Cell surface properties of organic solvent-tolerant mutants of Escherichia coli K-12.

In this study, we examined cell surface properties of mutants of Escherichia coli for which organic solvent tolerance levels were elevated. The cell surface of each mutant was less hydrophobic than that of the parent, probably due to an increase in lipopolysaccharide content. OmpF synthesis was repressed in the mutants. Organic solvent bound readily to viable E. coli cells in response to the polarity of the solvent. The mutants were bound less abundantly with the organic solvent than was the parent.

MicF antisense RNA has a major role in osmoregulation of OmpF in Escherichia coli.

micF RNA, produced from a multicopy plasmid, was originally shown to be a major factor in negative osmoregulation of the OmpF outer membrane protein in Escherichia coli. However, subsequent experiments with a micF deletion strain suggested that chromosomal micF RNA was not a key component in this process. We report here that micF RNA is essential for the reduction in OmpF levels in cells grown in media of low-to-intermediate levels of osmolarity. Under these conditions, the amount of OmpF was reduced up to 60% in the parent strain while OmpF levels were not altered in the micF deletion mutant. In medium of higher osmolarity, OmpF synthesis was strongly inhibited in both strains. RNA measurements showed that micF RNA levels rose rapidly in cells grown in low-to-intermediate levels of osmolarity concomitant with the reduction in OmpF protein, while ompF mRNA decreased strongly only during high-osmolarity conditions. Taken together, these results strongly suggest that the negative osmoregulation of OmpF at low-to-intermediate osmolarity levels requires micF RNA and that this is masked at higher osmolarity by the known strong inhibition of OmpF transcription by OmpR. Results consistent with this model were also obtained by using procaine, a compound reported to inhibit ompF expression by a mechanism very similar to that involved in osmoregulation.

Transient repression of the synthesis of OmpF and aspartate transcarbamoylase in Escherichia coli K12 as a response to pollutant stress

The synthesis of total cellular proteins in Escherichia coli K12 was studied in batch culture following exposure of cells to low concentrations of monochlorophenol, pentachlorophenol and cadmium chloride. Changes in protein patterns were identified after pulse-chase labelling of proteins with [35S]methionine and subsequent two-dimensional gel electrophoresis (2D-PAGE). We demonstrated that besides the induction of some stress proteins, also a transient decrease in the rate of synthesis of other proteins occurred. Two of these proteins were identified as OmpF and aspartate transcarbamoylase (ATCase). Their transient repression appeared to be a general response to stress elicited by different pollutants and may therefore be used as a general and sensitive early warning system for pollutant stress.

Transient repression of the synthesis of OmpF and aspartate transcarbamoylase inEscherichia coliK12 as a response to pollutant stress

The synthesis of total cellular proteins in Escherichia coli K12 was studied in batch culture following exposure of cells to low concentrations of monochlorophenol, pentachlorophenol and cadmium chloride. Changes in protein patterns were identified after pulse-chase labelling of proteins with [35S]methionine and subsequent two-dimensional gel electrophoresis (2D-PAGE). We demonstrated that besides the induction of some stress proteins, also a transient decrease in the rate of synthesis of other proteins occurred. Two of these proteins were identified as OmpF and aspartate transcarbamoylase (ATCase). Their transient repression appeared to be a general response to stress elicited by different pollutants and may therefore be used as a general and sensitive early warning system for pollutant stress.

Role of lipopolysaccharide in assembly of Escherichia coli outer membrane proteins OmpA, OmpC, and OmpF

Selection was performed for resistance to a phage, Ox2, specific for the Escherichia coli outer membrane protein OmpA, under conditions which excluded recovery of ompA mutants. All mutants analyzed produced normal quantities of OmpA, which was also normally assembled in the outer membrane. They had become essentially resistant to OmpC and OmpF-specific phages and synthesized these outer membrane porins at much reduced rates. The inhibition of synthesis acted at the level of translation. This was due to the presence of lipopolysaccharides (LPS) with defective core oligosaccharides. Cerulenin blocks fatty acid synthesis and therefore that of LPS. It also inhibits synthesis of OmpC and OmpF but not of OmpA (C. Bocquet-Pagès, C. Lazdunski, and A. Lazdunski, Eur. J. Biochem. 118:105-111, 1981). In the presence of the antibiotic, OmpA synthesis and membrane incorporation remained unaffected at a time when OmpC and OmpF synthesis had almost ceased. The similarity of these results with those obtained with the mutants suggests that normal porin synthesis is not only interfered with by production of mutant LPS but also requires de novo synthesis of LPS. Since synthesis and assembly of OmpA into the outer membrane was not affected in the mutants or in the presence of cerulenin, association of this protein with LPS appears to occur with outer membrane-located LPS.

The function of micF RNA: micF RNA is a major factor in the thermal regulation of OmpF protein in Escherichia coli

The role of chromosomally derived micF RNA as a repressor of outer membrane protein OmpF of Escherichia coli was examined for various growth conditions. Levels of micF RNA as determined by Northern analyses are found to increase in response to cell growth at high temperature, in high osmolarity or in the presence of ethanol. After a switch to higher growth temperature, the levels of ompF mRNA and of newly synthesized OmpF decrease with time in E. coli strain, MC4100 but these decreases are not observed in isogenic micF deletion strain, SM3001. In addition, while levels of ompF mRNA are substantially reduced in both strains in response to high osmolarity or ethanol at 24 degrees C, the reduced levels in the parental strain are still 4-5-fold lower compared with the micF deletion strain. These findings indicate that chromosomally derived micF RNA plays a major role in the thermal regulation of OmpF and represses OmpF synthesis in response to several environmental signals by decreasing the levels of ompF mRNA. Analyses of the effect of a multicopy micF plasmid on the levels of OmpF and ompF mRNA after an increase in temperature indicated that multicopies of micF RNA markedly inhibited OmpF synthesis but did not accentuate ompF mRNA decrease. These data suggest that multicopy micF inhibits OmpF synthesis primarily through translational inactivation of ompF mRNA and that a limiting factor in addition to micF RNA is necessary to destabilize ompF mRNA.

DNA-binding properties of the transcription activator (OmpR) for the upstream sequences of ompF in Escherichia coli are altered by envZ mutations and medium osmolarity.

Expression in Escherichia coli of the genes that encode the major outer membrane porin proteins (OmpF and OmpC) is regulated by the transcription activator protein OmpR and the receptorlike protein EnvZ, which is located in the inner membrane. Using synthesized oligonucleotide fragments containing the OmpR-binding site of ompF, we show that soluble extracts and partially purified OmpR derived from both the parent strain grown in nutrient broth plus 20% sucrose and the envZ11 strain grown in nutrient broth produced high-affinity DNA-binding activity, whereas soluble extracts from the parent strain grown in nutrient broth produced low-affinity binding. We also show that the soluble extracts from the envZ22(Am) strain grown in nutrient broth did not produce detectable bound forms of the ompF fragments, but low levels of DNA binding were detected with soluble extracts of the envZ22 strain grown in nutrient broth plus sucrose. In addition, the time course of the repression of OmpF synthesis produced by a shift to high-osmolarity growth medium was correlated with an increase in the DNA-binding affinity of soluble extracts to the ompF fragment. These results provide evidence that envZ function influences the DNA-binding activity of OmpR and suggest that high-affinity binding of OmpR to the upstream sequences of ompF is correlated with the repression of OmpF production.

The assembly of the major outer membrane protein OmpF of Escherichia coli depends on lipid synthesis.

Cerulenin, a drug which specifically blocks lipid synthesis, prevented both the trimerization of OmpF monomers and their assembly into the outer membrane of Escherichia coli B cells. A monoclonal antibody directed against a surface-exposed epitope of the trimer was used to probe the assembly of OmpF in the presence or absence of the drug. An inhibition level of 80% was reached 16 min after the addition of cerulenin. The accumulated monomeric form could not be assembled even after lipid synthesis was restored. Instead, it was slowly degraded. It was further shown that the inhibition of assembly resulted in a rapid inhibition of OmpF synthesis. These data demonstrate that there is a direct relationship between the synthesis of lipid (most likely lipopolysaccharide) and the correct export of OmpF. This coupling is required to promote the trimerization of the porin monomer and its assembly into the outer membrane.

Role of micF in the tolC-mediated regulation of OmpF, a major outer membrane protein of Escherichia coli K-12

Mutation in the tolC locus greatly reduces normal synthesis of OmpF, a major porin protein of Escherichia coli K-12. Experiments that use ompF-ompC chimeric genes demonstrate that a tolC mutation exerts its effect at either the promoter or the amino-terminal end of the ompF gene. Direct analysis of ompF mRNA from tolC+ and tolC strains showed that the amount of ompF transcript in the latter was greatly reduced. We have also observed that, in addition to reducing the amount of OmpF, a tolC mutation increases the level of OmpC protein to a much greater extent than occurs in an OmpF mutant and also increases micF RNA synthesis as shown by increased beta-galactosidase synthesis in a micF-lacZ fusion strain. Based on these observations, we suggest that an increased expression of the micF gene in a tolC mutant results in the reduced expression of ompF and that a major effect of the tolC mutation may be to push the porin-regulating system to favor ompC and micF to a greater extent than under high-osmolarity conditions.

Function of micF as an antisense RNA in osmoregulatory expression of the ompF gene in Escherichia coli

To analyze the function of micF as an antisera RNA in the osmoregulatory expression of the ompF gene in Escherichia coli, we performed two experiments. In the first experiment, two strains were constructed in which the transcription initiation site of the ompF gene and the transcription termination site of the micF gene were separated by 186 and 4,100 base pairs, respectively, on the chromosome. These two strains showed almost the same profile of ompF expression as the wild-type strain in which the two genes are separated by 10(6) base pairs. When a high-copy-number plasmid carrying the micF gene was introduced into these strains, ompF expression was completely repressed, whereas no repression was observed with a low-copy-number plasmid carrying the micF gene. These results indicate that the distance between the two genes on the chromosome is not critical for the function of micF. In the second experiment, expression of the ompF gene was examined by pulse-labeling in both the micF+ and the micF deletion strains. Upon a shift from a low- to a high-osmolarity medium, suppression of OmpF protein synthesis occurred more quickly and more extensively in the micF+ strain than in the micF deletion strain. The steady-state synthesis of the OmpF protein was also completely suppressed in the micF+ strain in the high-osmolarity medium, whereas the suppression was incomplete in the micF deletion strain. From these results we conclude that (i) the micF gene contributes to the fast and complete response of the OmpF synthesis to the medium osmolarity, and that (ii) the distance between the micF and ompF genes on the chromosomes is not critical for the function of the micF gene. The results suggest, rather, that the ratio of the copy numbers of the two genes is critical for the function of the micF gene.

Contrasting mechanisms of envZ control of mal and pho regulon genes in Escherichia coli.

The envZ11 missense mutation in the regulatory gene envZ pleiotropically repressed synthesis of OmpF, alkaline phosphatase, and several proteins of the maltose regulon. Procaine treatment of wild-type cells resulted in the same phenotype through an envZ+-mediated mechanism. Here we show that envZ11-procaine act differently on the mal and pho regulons. In the mal system, the expression of the positive regulator gene malT, measured as beta-galactosidase activity of a malT-lac+ operon fusion, was drastically reduced by procaine treatment or by the envZ11 mutation. In contrast, expression of the positive regulator of the pho regulon phoB was not reduced by procaine treatment. The products of the regulatory genes phoM, phoR, and phoU were also not required for procaine action. Procaine and envZ11 inhibited expression of only two products of the pho regulon, alkaline phosphatase and the PhoE porin. The conclusion that envZ11-procaine act differently on the mal and the pho regulons is supported by our ability to isolate second-site mutations with a Mal+ PhoA- phenotype in an envZ11 strain.

Construction and characterization of a deletion mutant lacking micF, a proposed regulatory gene for OmpF synthesis in Escherichia coli.

A method is presented for the construction of a deletion mutant lacking the micF gene, which has been proposed to negatively regulate expression of the ompF gene. The method includes (i) construction of a temperature-sensitive plasmid containing a chromosomal fragment that carries both flanking regions of the micF gene but does not carry micF itself and (ii) replacement of the corresponding chromosomal domain with the fragment. The method is applicable to construction of a deletion mutant for any Escherichia coli chromosomal gene provided that it is dispensable. The micF deletion was confirmed by genetic and biochemical tests, including nucleotide sequence analysis. ompF expression in the micF deletion mutant thus constructed was normally regulated and was not enhanced. When micF was cloned into a high-copy-number plasmid it repressed ompF gene expression, whereas when cloned into a low-copy-number plasmid it did not. From these results, it is concluded that a single copy of the micF gene on the E. coli chromosome does not play a critical role in ompF gene expression.

Mutation causing reverse osmoregulation of synthesis of OmpF, a major outer membrane protein of Escherichia coli

Supplementation of growth media with high concentrations of substances like sucrose results in the induction of OmpC synthesis and the suppression of OmpF synthesis. We isolated a novel mutant in which OmpF synthesis is in the opposite direction from normal osmoregulation. By transductional mapping, the mutation was localized at 75 min between malA and aroB on the Escherichia coli chromosome map where the ompR-envZ region is. The mutation was suppressed by a plasmid carrying the ompR gene but not by a plasmid carrying the envZ gene alone. The mutation also resulted in the almost complete suppression of OmpC synthesis. However, the remaining OmpC synthesis was osmoregulated normally. Based on these observations, the mechanism of osmoregulation of OmpF-OmpC synthesis is discussed.

Regulation of outer membrane protein synthesis in Escherichia coli K-12: deletion of ompC affects expression of the OmpF protein.

A chromosomal deletion beginning at a Tn10 located ca. 8 kilobases upstream from the ompC structural gene and extending through the 2.6-kilobase HindIII fragment carrying the ompC was isolated. The 2.6-kilobase ompC fragment was cloned into lambda 540 to obtain phage lambda 540C1. When the deletion mutant was lysogenized with lambda 540C1, the resulting strain produced normal levels of OmpC protein, and expression of this protein was regulated by osmolarity, carbon source, and the lc gene of phage PA-2, indicating that the cloned fragment contained all of the information required for regulated expression of ompC. The strain carrying the deletion was partially constitutive for expression of OmpF protein, whereas the lambda 540C1 lysogen of this strain and other strains with mutations in ompC repressed OmpF synthesis under conditions which lead to high-level expression of OmpC protein. Strains which are diploid or triploid for ompC show strong inhibition of synthesis of OmpF protein. We conclude that a regulatory element located upstream from the ompC coding sequence inhibits translation of OmpF protein under conditions which favor OmpC expression. Since ompF is known to repress transcription of ompC, we propose that these two genes constitute a closed regulatory loop which acts to amplify regulatory signals which control expression of these proteins.

Mutation causing overproduction of outer membrane protein OmpF and suppression of OmpC synthesis in Escherichia coli.

A novel mutation affecting the synthesis of major outer membrane proteins OmpF and OmpC in Escherichia coli K-12 is described. The mutation resulted in overproduction of the OmpF protein with concomitant suppression of OmpC synthesis. This mutation, designated as ompFp100, was mapped at 21 min on the E. coli chromosome map with the gene order aroA-aspC-ompF4-ompFp100-asnS-pyrD. This mutation was cis-dominant to the expression of the ompF gene. In addition, the direction of the mRNA transcription of the ompF gene was from asnS to aspC. These results strongly indicate that ompFp100 is a promoter mutation of the ompF gene. Introduction of an ompF mutation, which causes the disappearance of the OmpF protein, into strains carrying the ompFp100 mutation resulted in the reappearance of the OmpC protein in the outer membrane. Addition of a high concentration of sucrose to the medium, which suppresses the OmpF synthesis and stimulates the OmpC synthesis in the wild-type strain, resulted in the reappearance of the OmpC protein in the ompFp100 mutant with concomitant suppression of the overproduction of the OmpF protein. These results suggest that suppression of OmpC synthesis in the ompFp100 mutant is due to overproduction of the OmpF protein.

Regulation of outer membrane porin protein synthesis in Escherichia coli K-12: ompF regulates the expression of ompC.

The relative amounts of the OmpF and OmpC proteins in the outer membrane of Escherichia coli K-12 are affected differentially by high concentrations of substances like sucrose in culture media in such a manner that a decrease in the amount of the OmpF protein appears to be compensated for by a reciprocal increase in the OmpC protein. When an ompF mutation was introduced, OmpC synthesis became almost independent of sucrose and occurred at the fully induced level even in the absence of sucrose. On the other hand, introduction of an ompC mutation did not affect the sucrose-dependent profile of OmpF synthesis. The effect of the ompF mutation was also examined with the ompC-lac fusion strain, in which expression of beta-galactosidase is under the control of the ompC promoter. The expression of beta-galactosidase coded for by the ompC-lac fusion in the ompF+ and ompF- strains was essentially the same as that of the OmpC protein, being sucrose dependent in the ompF+ strain and sucrose-independent in the ompF mutant. From these results we conclude that sucrose in the medium primarily regulates ompF gene expression, which in turn regulates ompC gene expression at the transcriptional level. This sequential regulatory mechanism is discussed in relation to the function of the ompB locus.